Staphylococcus aureus is an opportunistic pathogen that produces a diverse array of virulence factors and causes a correspondingly diverse array of infections. Our long-term goal is to elucidate the regulatory mechanisms controlling expression of these virulence factors as a necessary prerequisite to the development of therapeutic protocols capable of attenuating the disease process. The specific hypothesis is that the staphylococcal accessory regulator (sar) is a major regulatory locus controlling expression of S. aureus virulence factors. We base that hypothesis on the observations that 1) sar encodes a DNA-binding protein (SarA) required for expression of the agr-encoded, RNAIII regulatory molecule, 2) phenotypic comparison os sar and agr mutants demonstrates that sar also regulates expression of S. aureus virulence factors in an agr-independent manner and 3) mutation of sar and agr results in reduced virulence even by comparison to agr mutants. Based on these observations, the experimental focus of this proposal is on the agr-independent branch of the sar regulatory pathway. The specific aims are to: 1. Define the relationship between sar transcription and the production of functional SarA. We will correlate the production of the SarA, SarB, and SarC transcripts with (i) the production of SarA, (ii) the DNA-binding activity of SarA and (iii) the ability of SarA to regulate transcription of a target gene. 2. Characterize the mechanisms of sar-mediated regulation of cna transcription. The S aureus collagen adhesin gene (cna) is expressed in a growth-phase dependent manner and that sar is the primary regulatory element controlling cna transcription. Preliminary experiments indicate that the regulatory impact of sar on cna transcription involves a direct interaction between SarA and DNA upstream of cna. We will identify the sar transcripts required to complement the cna defect and will correlate the results of our complementation studies with the production and activity of SarA. We will also identify and characterize the cis elements that define cna as a target for sar-mediated regulation. 3. Identify S. aureus genes under the direct regulatory control of SarA. We will (i) characterize the consensus SarA-binding site, (ii) identify putative SarA targets within the S. aureus genome and (iii) confirm the sar-mediated regulation of these targets by Northern blot analysis of sar mutants.